Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic " Lab-on-a-Disc " cartridge. Isolation of white blood...
moreHere we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic " Lab-on-a-Disc " cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are 'low-pass', i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both 'hydrostatically' and 'hydrodynamically' triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, 'dual siphon' configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is character-ised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction.
There is increasing evidence that, in addition to their presence, the propensity of circulating tumour cells to form multi-cellular clusters bears significant information about both cellular resistance to chemotherapy and overall...
moreThere is increasing evidence that, in addition to their presence, the propensity of circulating tumour cells to form multi-cellular clusters bears significant information about both cellular resistance to chemotherapy and overall prognosis. We present a novel two-stage, stopped-flow, continuous centrifugal sedimentation strategy to measure the size distributions of events (defined here as cells or clusters thereof) in a blood sample. After off-chip removal of red blood cells, healthy white blood cells are sequestered by negative-immunocapture. The purified events are then resolved along a radially inclined rail featuring a series of gaps with increasing width, each connected to a designated outer collection bin. The isolation of candidate events independent of target-specific epitopes is successfully demonstrated for HL60 (EpCAM positive) and sk-mel28 (EpCAM negative) cells using identical protocols and reagents. The propensity to form clusters was quantified for a number of cell lines, showing a negligible, moderate or elevated tendency towards cluster formation. We show that the occupancy distribution of the collection bins closely correlates with the range of cluster sizes intrinsic to the specific cell line.
Even though an agreed phenotypic definition of circulating tumor cells (CTCs) remains elusive in the literature, many current detection technologies isolate candidate cells based on molecular recognition of cellular epitopes that may not...
moreEven though an agreed phenotypic definition of circulating tumor cells (CTCs) remains elusive in the literature, many current detection technologies isolate candidate cells based on molecular recognition of cellular epitopes that may not accurately predict CTC load. Rather than using such an epitope specific "positive-capture" strategy, we present a chip-based, centrifugal microfluidic platform integrating "negative-capture" magnetophoretic removal of normal white blood cells (WBCs) from a sample and subsequent, array-based enumeration of individualized, (untagged) abnormal cells. We compared the numerical recovery of cells on the array with the status of the donor patient, showing that the chip can has the potential to indicate the oncogenic severity of the blood donor. INTRODUCTION With advances in modern therapies, the prognosis for patients presenting with cancer can be positive in cases where malignancies are detected early. The detection and / or enumeratio...
We present a substantially improved design and functionality of a centrifugo-magnetophoretic platform which integrates direct immunoseparation and cost-efficient, bright-field detection of cancer cells in whole blood. All liquid handling...
moreWe present a substantially improved design and functionality of a centrifugo-magnetophoretic platform which integrates direct immunoseparation and cost-efficient, bright-field detection of cancer cells in whole blood. All liquid handling takes place in a disposable cartridge with geometry akin to a conventional compact disc (CD). The instrumentation required to process such a "lab-on-a-disc" cartridge can be as simple and cost-efficient as the rotor on a common optical disc drive. In a first step, target cells in a blood sample are specifically bound to paramagnetic microbeads. The sample is then placed into the disc cartridge and spun. In the second step, magnetically tagged target cells are separated by a co-rotating, essentially lateral magnetic field from the background population of abundant blood cells, and also from unbound magnetic beads. A stream of target cells centrifugally sediments through a stagnant liquid phase into a designated detection chamber. The contin...
ABSTRACT This paper reports radially inbound pumping by the event-triggered addition of water to on-board stored baking powder in combination with valving by an immiscible, high-specific weight liquid on a centrifugal microfluidic...
moreABSTRACT This paper reports radially inbound pumping by the event-triggered addition of water to on-board stored baking powder in combination with valving by an immiscible, high-specific weight liquid on a centrifugal microfluidic platform. This technology allows making efficient use of precious real estate near the center of rotation by enabling the placement of early sample preparation steps as well as reagent reservoirs at the spacious, high-field region on the perimeter of the disc-shaped rotor. This way the number of process steps and assays that can be integrated on these of this " Lab-on-a-Disc " (LoaD) cartridge can be significantly enhanced while maintaining minimum requirements on the intrinsically simple, spindle-motor based instrumentation.
ABSTRACT We integrate microfluidic and label-free optical technologies with highly efficient cell-to-light coupling to measure the real-time response of individual cells in a population to specific stimuli. The innate optical response of...
moreABSTRACT We integrate microfluidic and label-free optical technologies with highly efficient cell-to-light coupling to measure the real-time response of individual cells in a population to specific stimuli. The innate optical response of the cell comprises of scattering, absorbance and auto-fluorescence signals as well as cell morphology. We demonstrate that the so-obtained “photonic fingerprint” acts as a key reporter for the dysfunction of cells and may thus inform on a patient’s medical condition. To perform analysis at single cell level, our centrifugal microfluidic “lab-on-a-disc” platform features a V-cup array geared for cell capture at a sharply peaked single-occupancy distribution. To support the formal process of obtaining ethical approval for access to patient samples, we demonstrate functionality on commercial human umbilical vein endothelial cells (HUVECs) and endothelial somatic cell hybrids. Differential inflammation has been induced by exposing the cells to various concentrations of protein tumour necrosis factor alpha during culture. The developed, label-free biophotonic system obtains a specific and unique photonic signal for each cell condition investigated. The level of cell dysfunction can be classified based on the obtained photonic fingerprint. The key advantage of this configuration is a highly efficient cell-to-light interaction, enabling the end user to detect the presence of diseases by measuring the cell photonic fingerprint
The faithful replication of DNA and the accurate segregation of genomic material from one generation to the next is critical in the maintenance of genomic stability. This chapter will describe the structure and assembly of an...
moreThe faithful replication of DNA and the accurate segregation of genomic material from one generation to the next is critical in the maintenance of genomic stability. This chapter will describe the structure and assembly of an epigenetically inherited locus, the centromere, and its role in the processes by which sister chromatids are evenly segregated to daughter cells. During the G2 phase of the cell cycle kinetochores are assembled upon the chromatids. During mitosis, kinetochores attach chromosome(s) to the mitotic spindle. The kinetochore structure serves as the interface between the mitotic spindle and the chromatids and it is at the kinetochore where the forces that drive chromatid separation are generated. Unattached chromosomes fail to satisfy the spindle assembly checkpoint (SAC), resulting in cell cycle arrest. The centromere is the locus upon which the kinetochore assembles, and centromeres themselves are determined by their unique protein composition. Apart from budding yeast, centromeres are not specified simply by DNA sequence, but rather through chromatin composition and architecture and are thus epigenetically determined. Centromeres are built on a specific nucleosome not found elsewhere in the genome, in which histone H3 is replaced with a homologue - CENP-A or CenH3. This domain is flanked by heterochromatin and is folded to provide a 3-dimensional cylinder-like structure at metaphase that establishes the kinetochore on the surface of the mitotic chromosomes. A large family of CENtromere Proteins (CENPs) associates with centromeric chromatin throughout the cell cycle and are required for kinetochore function. Unlike the bulk of histones, CENP-A is not assembled concurrently with DNA synthesis in S-phase but rather assembles into the centromere in the subsequent G1 phase. The assembly of CENP-A chromatin following DNA replication and the re-establishment of this network of constitutive proteins have emerged as critical mechanisms for understanding how the centromere is replicated during the cell cycle.
ABSTRACT This work reports a novel, versatile, and membrane-integrated 3-dimensional microfluidic platform (MP) for high-throughput biodiagnostics, with an aim towards applications in clinical cancer studies. The hybrid MP is designed to...
moreABSTRACT This work reports a novel, versatile, and membrane-integrated 3-dimensional microfluidic platform (MP) for high-throughput biodiagnostics, with an aim towards applications in clinical cancer studies. The hybrid MP is designed to be compatible with standard equipment used in high throughput screening, featuring membrane supported micro-wells and gradient generating microstructures to enable exposure of cell samples to various concentrations of reagents of interest, suitable to run both fluorometric and colorimetric assays. This paper discusses design and fabrication of the MP, and also describes preliminary testing of colorimetric and fluorometric assays carried out on the device.
ABSTRACT We present a disc-based module for rotationally controlled solid-phase purification of RNA from cell lysate. To this end, multi-stage routing of a sequence of aqueous and organic liquids into designated waste and elution...
moreABSTRACT We present a disc-based module for rotationally controlled solid-phase purification of RNA from cell lysate. To this end, multi-stage routing of a sequence of aqueous and organic liquids into designated waste and elution reservoirs is implemented by a network of strategically placed, solvent-selective composite valves . Using a bead-based stationary phase at the entrance of the router, we show that total RNA is purified with high integrity from cultured MCF7 and T47D cell lines, human leucocytes and Haemophilus influenzae cell lysates. Furthermore, we demonstrate the broad applicability of the device through the in vitro amplification of RNA purified on-disc using RT-PCR and NASBA. Our novel router will be at the pivot of a forthcoming, fully integrated and automated sample preparation system for RNA-based analysis .
Modern advancements in pharmaceuticals have provided individuals who have been infected with the human immunodeficiency virus (HIV) with the possibility of significantly extending their survival rates. When administered sufficiently soon...
moreModern advancements in pharmaceuticals have provided individuals who have been infected with the human immunodeficiency virus (HIV) with the possibility of significantly extending their survival rates. When administered sufficiently soon after infection, antiretroviral therapy (ART) allows medical practitioners to control onset of the symptoms of the associated acquired immune deficiency syndrome (AIDS). Active monitoring of the immune system in both HIV patients and individuals who are regarded as "at-risk" is critical in the decision making process for when to start a patient on ART. A reliable and common method for such monitoring is to observe any decline in the number of CD4 expressing T-helper cells in the blood of a patient. However, the technology, expertise, infrastructure and costs to carry out such a diagnostic cannot be handled by medical services in resource-poor regions where HIV is endemic. Addressing this shortfall, commercialized point-of-care (POC) CD4 cell count systems are now available in such regions. A number of newer devices will also soon be on the market, some the result of recent maturing of charity-funded initiatives. Many of the current and imminent devices are enabled by microfluidic solutions, and this review will critically survey and analyze these POC technologies for CD4 counting, both on-market and near-to-market deployment. Additionally, promising technologies under development that may usher in a new generation of devices will be presented.
The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in RAD30, which encodes the novel lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and...
moreThe human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in RAD30, which encodes the novel lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and increased mutagenesis following UV irradiation. In cell-free extracts of XPV lymphoblasts, functional DNA polymerase eta is required for the complete replication of a double-stranded plasmid containing either a single (6-4) photoproduct or a cyclobutane pyrimidine dime(CPD), the major mutagenic UV-induced lesion. In cultured XPV cells, replication arrest activates downstream signalling pathways, leading to hyperphosphorylation of the 34-kDa subunit of the trimeric single-stranded DNA-binding protein, RPA (replication protein A). Many of the RAD30 mutations identified in XPV cells result in truncation and inactivation of DNA polymerase eta. To examine whether polymorphisms in the RAD30 gene that result in altered polymerase eta function, rather than enzyme inactivation, might contribute to individual susceptibility to skin cancer, methods to screen for sequence changes in the RAD30 gene in human genomic DNAhave been developed.